This invention relates to emulsions and their use as delivery vehicles for biologically active molecules. In particular, the invention relates to emulsion formulations comprising an aqueous carrier and the following components: triglyceride, cholesterol, phospholipid, charged lipid, hydrophilic biologically active molecule and optionally, cholesteryl ester, and/or apoprotein(s); to methods of preparing these emulsions; and to their uses as vehicles for the delivery of hydrophilic biologically active molecules to cells.
The field of selective delivery of biologically active molecules to specific cellular targets is rapidly advancing. Among the various systems that have been studied for their ability to deliver bioactive molecules to cells, lipoproteins, which form naturally occurring biological emulsions, are considered attractive as delivery vehicles for a number of reasons: 1) as endogenous carriers of cholesterol and other lipids in the blood circulation, they are not immunogenic and escape recognition by the reticuloendothelial system; 2) physicochemical characterization of drug-loaded lipoproteins indicates that particles with the same physicochemical properties as the native lipoproteins can be obtained; 3) lipoproteins are removed from the circulation by specific receptors that recognize their apoproteins or, they may be directed to nonlipoprotein receptors by chemical modification of their apoproteins (see, for example, Bijsterbosch, M. K. and van Berkel, T. J. C. (1990) Adv. Drug Delivery Revs, 5:231-251); 4) lipoproteins are physically stable due to their compact, neutral, apolar core; 5) the apolar core of lipoproteins provides an ideal domain for lipophilic molecules since molecules that are transported in the core of the lipoprotein are protected from the environment during transportation and the environment is protected from the molecule; and 6) lipoproteins can be synthesized from commercially available lipids and isolated apoproteins.
Examples of the use of lipoproteins as delivery vehicles for bioactive molecules include U.S. Pat. Nos. 4,868,158 and 5,324,821 which refer to the preparation of lipoproteins modified by incorporation of a lipophilic active substance into their apolar core. However, since many of the biologically active molecules used for treatment and/or prevention of diseases are too hydrophilic for incorporation into the apolar core of lipoproteins, investigators have attempted to find methods which would permit the incorporation of hydrophilic molecules into lipoproteins.
One approach that has been utilized is to couple hydrophilic molecules to hydrophobic anchors in order to render the hydrophilic molecule more lipophilic. For example, van Berkel has reported the synthesis of a dioleoyl derivative of the anti-viral nucleoside analogue iododeoxyuridine and its incorporation into either high density lipoproteins (HDL) (Bijsterbosch, M. K. et al. (1994) Biochemistry, 33:14073-14080) or chylomicrons (Rensen, P. C. N. et al. (1995) Nature Medicine, 1:221-225). Unfortunately, the use of conjugation to facilitate incorporation of hydrophilic molecules into lipoproteins has a number of potential drawbacks: 1) since the conjugated molecules are typically incorporated into a lipid or a protein residue on the surface of the lipoproteins, the conjugated molecule may interfere with the interaction of the apoprotein of the lipoprotein and its receptor; 2) such surface-modified particles may show a greater tendency to aggregate due to a potential loss of surface charge; 3) attachment of bioactive molecules to the surface of lipoproteins exposes the molecules to the environment and vice versa; 4) xe2x80x9cderivatizationxe2x80x9d of hydrophilic molecules by conjugation to hydrophobic anchors may affect the biological activity of the hydrophilic molecule; and 5) such a xe2x80x9cconjugationxe2x80x9d approach is impractical for rendering larger molecular weight hydrophilic molecules hydrophobic enough to be incorporated into the apolar core of the lipoproteins.
The present invention relates to emulsion formulations designed to mimic native (i.e. naturally occurring) lipoprotein emulsions in vivo.
The emulsion formulations of the invention comprise an aqueous carrier and the following components: triglyceride, cholesterol, phospholipid, at lease one charged lipid, at least one hydrophilic biologically active molecule, and optionally, protein(s), and/or cholesteryl ester. By xe2x80x9cproteinxe2x80x9d is meant a protein that targets the emulsion to a specific cell type. A preferred protein is an apoprotein, where the apoprotein may be free of lipid or may be complexed covalently or ionically with lipid to form an apolipoprotein.
In formulating the emulsions of the invention, the charged lipid and hydrophilic biologically active molecule are first mixed together in a charged lipid: bioactive molecule ratio suitable to result in the formation of a hydrophobic charged lipid:bioactive molecule complex (herein xe2x80x9chydrophobic complexxe2x80x9d) which is soluble in the organic phase of a two phase aqueous-organic system. This hydrophobic complex is then mixed with triglyceride, cholesterol, phospholipid and optionally, cholesteryl ester, and/or apoprotein, to form the emulsions of the invention.
In a preferred embodiment, at least one negatively charged bioactive molecule is mixed together with at least one cationic lipid to form the hydrophobic complex.
In an alternative embodiment at least one positively charged bioactive molecule may be mixed together with at least one anionic lipid to form the hydrophobic complex.
By cationic lipid or anionic lipid is meant a lipid having a net positive (cationic lipid) or net negative (anionic lipid) charge at or near physiological pH.
It is believed that the emulsions of the present invention therefore comprise an apolar core composed of triglycerides and optionally, cholesteryl esters, into which the hydrophobic complex of a hydrophilic bioactive molecule and preferably, a cationic lipid, is incorporated; the apolar core being surrounded by a phospholipid monolayer in which cholesterol and optionally, specific apoprotein(s) are incorporated.
The invention also relates to methods of producing the emulsion formulations of the invention.
In one embodiment, the emulsion formulation of the invention may be produced by:
(a) mixing a hydrophobic complex of hydrophilic bioactive substance and cationic lipid with triglyceride, phospholipid, cholesterol and optionally cholesteryl ester in an organic solvent;
(b) removing the organic solvent to leave a lipid film;
(c) and suspending the film in an aqueous buffer to produce the emulsion.
If the emulsions are not uniform in size, the emulsions may be further purified to remove emulsions of undesired size. Such purification also serves to sterilize the emulsions.
In an alternative embodiment, where the emulsion formulation of the invention is to contain a water-soluble apoprotein, the apoprotein may be added to, and mixed with, the emulsion formed by the suspension of the film in step (c) above.
Of course, when isolated apoproteins such as apo-B-48 and apo-B-100, which are not soluble in aqueous buffer, are to be included in the emulsions, the above method of producing an emulsion containing apoproteins may be modified such that apoprotein is included among the components combined in the organic solvent prior to the film formation.
In an alternative embodiment, the emulsion formulations of the invention may be produced by dissolving the hydrophobic complex of cationic lipid and a hydrophilic bioactive molecule in the apolar core of a reconstituted lipoprotein emulsion.
In one embodiment, such a method comprises:
(a) extracting native lipoprotein, which preferably has been lyophilized, with an organic solvent;
(b) mixing the extracted lipoprotein lipids with a hydrophobic complex of cationic lipid and hydrophilic bioactive molecule in the organic solvent;
(c) removing the organic solvent to leave a lipid film;
(d) adding aqueous carrier to produce the emulsion; and
(e) purifying said emulsion, if necessary, to sterilize and obtain emulsions of desired size.
Typically, the emulsion formulations produced by this method would be apoprotein-free emulsions since most apoproteins are water-soluble and would not be contained in the organic solvent extract.
However, as noted above, apoproteins such as xcex2-48 and xcex2-100 are extractable with organic solvent. Therefore, it is understood that in cases where the lipoprotein to be extracted in step (a) is, for example, LDL, the organic extract to be mixed with hydrophobic complex in step (b) would contain apo-B-100 as well as the lipoprotein lipids. Of course, it is understood that even if the lipoprotein selected to be extracted by organic solvent does not contain apoprotein soluble in organic solvent, water-soluble apoprotein can be added to the aqueous suspension produced in step (d) in the above method to produce an apoprotein-containing emulsion.
In yet another embodiment, the emulsions of the invention may be produced by preparing the hydrophobic complex of cationic lipid and hydrophilic biologically active molecule as a dry film (i.e. by removal of the organic solvent into which the hydrophobic complex partitions), then adding native lipoproteins to the dry film and vortexing, such that the hydrophobic complex contained in the dry film rehydrates and partitions into the apolar core of the lipoprotein.
The invention therefore also relates to a lipid film capable of forming the emulsion formulations of the present invention upon suspension in an aqueous carrier. The film comprises triglyceride, cholesterol, phospholipid, cationic lipid, hydrophilic biologically active molecule and optionally, cholesteryl ester and/or water-insoluble apoproteins. Where the film comprises apoprotein, it is designated a lipid protein film.
The invention also relates to methods for delivering hydrophilic bioactive molecules to cells in vitro or in vivo.
In one embodiment, the method of delivering hydrophilic biologically active molecules to cells in vivo comprises administering to an animal an effective amount of an emulsion formulation of the present invention. Preferred animals are humans. Thus, for example, where the bioactive molecule is a gene or an oligonucleotide, the invention relates to the use of the emulsion formulations in gene or oligonucleotide therapies respectively.